Bhb colorimetric assay kit

BHB and blood glucose concentrations were measured using the Precision Xtra and ReliOn Confirm systems, respectively. Urine ketone levels were qualitatively evaluated using Ketostix reagent strips for urinalysis (Bayer, Leverkusen, Germany). Skeletal muscle tissue BHB levels were measured using the BHB colorimetric assay kit (k632-100, Bio Vision, Milpitas, CA). The serum were obtained from blood in blood collecting tubes (BD bioscience) following centrifuge as manufacture’s protocol when the mice were euthanized. The serum insulin levels were measured using an ELISA (ALPCO, Windham, NH). Serum cholesterol levels were measured using a cholesterol assay kit (ab65390, Abcam), and serum triglyceride levels were measured using a triglyceride colorimetric assay kit (#10010303, Cayman Chemical, Ann Arbor, MI).

The cells seeded in 12-well plates were treated with DMSO (control) or fenofibrate and/or MK886 for 48 h. The media were collected and centrifuged to eliminate any possible cell debris and then processed immediately or frozen in −80°C. The concentration of bHB in the cell culture media aliquots of 50 μL was measured using bHB colorimetric assay kit (Biovision #K632, USA), according to the manufacturer’s instructions. The bHB concentrations were calculated from the 450 nm absorbance recorded from the microplate reader and the standard curve prepared with the 1 mM bHB solution included in the kit. Every time the bHB production was normalized to the total cell number in each well. For this purpose and for assessment of cell proliferation, cells were fixed and stained with crystal violet solution, and the cell numbers were determined by a spectrophotometric method, according to Kueng et al. (46 (link)).